1 One of the goals of systems neuroscience is to understand how sensory information is transformed into goal- 2 directed behavior via diverse brain regions and circuits. To achieve this aim, it is critical to elucidate computations 3 performed within specific layers of the cortex by specific cell classes and the communication dynamics between 4 multiple brain regions. Two-photon microscopy has been used successfully to perform functional brain imaging 5 at the single-cell level mice, but its penetration is limited by tissue scattering to the top layers of the cortex. I have 6 developed a 3-photon microscope to overcome this challenge. Today, the main drawback of 3-photon 7 microscope is its relatively modest speed, limiting its use for multi-site imaging. Optimizing instrument design 8 and imaging protocol to overcome this limitation is required for broad end-user acceptance. In this proposal, I 9 will construct and optimize a combined 2-photon and 3-photon microscope for multi-site, superficial and deep 10 brain imaging at single-cell resolution. Specifically, I have first developed a custom-made 3-photon microscope 11 with optimized laser and microscope parameters (Aim 1a). Optimizing these parameters can improve imaging 12 speed and imaging depth while lowering the average laser power to avoid damage in the live mouse brain. The 13 microscope performance improvement has been validated by performing functional imaging in the primary visual 14 cortex of GCaMP6 mice to characterize visual responses of each cortical layer and subplate. In addition, I will 15 characterize the effective attenuation lengths (EAL) of higher visual areas in awake mice with label-free imaging 16 and laser-ablation methods. Then, I will demonstrate the microscope?s performance by examining cell-specific 17 differences within a layer 6 (L6) of V1. Since neuronal responses to visual stimuli are modulated by the cortical 18 state such as arousal, or reward expectation, I will image adjacent sets of neurons with distinct projections to the 19 lateral geniculate nucleus (LGN) and lateral posterior (LP) regions (e.g., cortico-cortical [CC] and cortico-thalamic 20 [CT] neurons in L6) in primary and higher visual areas to reveal circuit-based response types within a single 21 cortical layer using retrobead-based tracing methods (Aim 1b). Next, I have developed custom-made 2-photon 22 wide-field microscope to perform neuronal recordings and manipulations in the primary visual cortex and higher 23 visual areas (Aim 2a). I have improved imaging speed and field of view by implementing multifocal multiphoton 24 microscopy (MMM). Multiple foci two-photon excitation efficiency will be optimized by coupling a diffractive 25 element (DOE) with customized intermediate optics. High sensitivity single-photon counting detection will be 26 achieved using a novel avalanche photodiode array detector. To demonstrate microscope performance and 27 which brain regions are necessary for a well-established goal-directed behavioral paradigm, I will perform SLM- 28 based two-photon optogenetics while imaging expert animals (Aim 2b). In addition to imaging and stimulating 29 neuronal activity across superficial depths at single regions and at multiple regions, it is necessary to image and 30 optogenetically manipulate neuronal activity at multiple depths, at targeted locations, and for identified neurons, 31 in order to determine the causality of neuronal subpopulations in behavior. Here, I will design and implement 32 two- and three-photon MMM systems to extend the depth performance of MMM for multi-site neuronal recording 33 across multiple regions and multiple layers and integrate this system with the 2-photon optogenetics system 34 implemented in Aim 2a (Aim 3a). I will use this technology for modulating specific components of the cortico- 35 cortical and cortico-thalamo-cortical projections of V1-V2-PPC-MC circuit (Aim 3b).